Assay Overview: The assay uses A549 cells obtained from ATCC to test for cytotoxicity at the same concentrations used to confirm compound activity in the primary screen. Cellular ATP levels are measured as a surrogate of cell viability with the luminescence reagent, CellTiter-Glo (Promega). ..more

Coupled Fluorescence Displacement Counter Screen for interference with FL-DTB/streptavidin and against BioD Measured in Biochemical System Using Plate Reader - 2163-13_Inhibitor_Dose_DryPowder_Activity

Assay Overview: The assay uses A549 cells obtained from ATCC to test for cytotoxicity at the same concentrations used to confirm compound activity in the primary screen. Cellular ATP levels are measured as a surrogate of cell viability with the luminescence reagent, CellTiter-Glo (Promega).

Expected Outcome: Compounds identified as hits will be toxic to cells at a compound concentration less than 10 uM. Activity in the assay leads to a reduction in cellular ATP levels which correlates with a decreased luminescence signal from the read reagent (Promega's CellTiter-Glo) and indicates cytotoxicity. Compounds that exhibit no cytotoxicity at an IC50 >30 uM will be prioritized for additional studies.

PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) and positive control wells (PC; n=32) were included on every plate.EXPECTED OUTCOME: Active compounds result in decreasing readout signal.ACTIVE CONCENTRATION LIMIT:For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).NORMALIZATION:The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.Experimental wells values were scaled to this range.PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): absACnn, the concentration at which the curve crosses threshold -50.0AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).AC values were calculated up to the active concentration limit described for each sample.pAC was set to equal -1*log10(AC)PUBCHEM_ACTIVITY_OUTCOME:Activity_Outcome = 1 (inactive) when:a) compound shows activity but in a direction opposite to the expected outcomein these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to nullb) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, orc) AC > AC_limitActivity_Outcome = 2 (active) when:AC <= AC_limitActivity_Outcome = 3 (inconclusive) when:a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, orb) The fit was deemed not valid due to poor fit quality.PUBCHEM_ACTIVITY_SCORE:If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),then PUBCHEM_ACTIVITY_SCORE = 0If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)Scores relate to AC in this manner:120 = 1 pM90 = 1 nM60 = 1 uM30 = 1 mM0 = 1 MWhen the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.Note:The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.